ABSTRACT There are high prevalences of osteoporotic fractures and vitamin D deficiency in the U.S., especially among older adults. Although vitamin D supplements are widely used to improve bone health, evidence for a role of supplemental vitamin D alone in reducing fractures is lacking. While the serum 25-hydroxyvitamin D [25(OH)D] level is considered the best analyte to assess vitamin D status, currently there is no consensus on the optimal circulating total 25(OH)D concentration for bone. While, like many hormones, vitamin D circulates bound to proteins, the relative importance of free 25(OH)D (FVD) or bioavailable 25(OH)D levels on fracture risk is not known. New advances in technology make it possible to directly measure the biologically active, FVD level. Results from recent observational studies on whether FVD and/or bioavailable 25(OH)D are more strongly associated than total 25(OH)D levels with calcium homeostasis and bone mineral density (BMD) are inconsistent. In addition, there are no data from large, randomized controlled trials on whether bioavailable 25(OH)D and/or FVD vs. total 25(OH)D levels or gene variations in vitamin D-related pathways modify effects of supplemental vitamin D on fractures and changes in BMD. To fill gaps in knowledge, we propose an ancillary study to the large, NIH-sponsored, randomized, controlled VITamin D and OmegA-3 TriaL (VITAL) that is testing effects of supplemental vitamin D3 (cholecalciferol, 2000 IU/d), and/or omega-3 fatty acids (fish oil, 1 g/d) in the primary prevention of cancer and cardiovascular disease in 25,874 U.S. men (aged ?50) and women (aged ?55), including 5,107 African Americans. The proposed ancillary study will definitively determine whether supplemental, high-dose, vitamin D3 alone reduces incident total, non-vertebral and hip fractures by extending adjudication of fractures to the full 5 years of treatment in 25,874 VITAL participants nationwide. The proposed studies will also rigorously test whether concentrations of bioavailable 25(OH)D and/or FVD are more strongly associated with changes in BMD and incident fractures than total 25(OH)D levels; and whether these vitamin D biomarkers and genetic variants in vitamin D-related pathways modify effects of supplemental vitamin D on BMD and fracture risk. For each of the proposed aims, we will assess whether results vary by (a) sex, (b) race/ethnicity, and (c) BMI. Key VITAL resources will be leveraged at no additional cost to the proposed studies including: blood samples and BMD measurements (at baseline and follow-up), extensive data on changes in 25(OH)D levels over time (in the treatment vs. placebo arms), measures of calcium homeostasis, information on clinical risk factors, and extracted DNA. This proposal provides a unique and cost- efficient opportunity to generate important positive or informative negative results about effects of supplemental vitamin D3 alone on fracture risk, while also elucidating the relative importance of vitamin D biomarkers and genetic variations in vitamin D-related pathways on bone. Findings from the proposed ancillary study have the potential for major clinical as well as public health impact for both men and women in the U.S.